Several scientific or patent publications are referenced in this patent application to describe the state of the art to which the invention pertains. Each of these publications is incorporated by reference herein, in its entirety.
Mammals respond to tissue injury, trauma or infection by executing a complex series of biological reactions in an effort to prevent further tissue damage, to initiate repair of damaged tissue, and to isolate and destroy infective organisms. This process is referred to as the inflammatory response, the early and intermediate stages of which are referred to as the acute phase response.
The acute phase response involves a wide variety of mediators, including cytokines, interleukins and tumor necrosis factor. It also involves a radical alteration in the biosynthetic profile of the liver. Under normal circumstances, the liver synthesizes a range of plasma proteins at steady state concentrations. Some of these proteins, the “acute phase” proteins are induced in the inflammatory response to a level many times greater than levels found under normal conditions. Acute phase proteins are reviewed by Steel & Whitehead (Immunology Today 15: 81-87, 1994).
One of the massively induced acute phase proteins is serum amyloid A (SAA). SAAs are small apolipoproteins that accumulate and associate rapidly with high-density lipoprotein 3 (HDL3) during the acute phase of the inflammatory response. Most SAA isoforms are induced in response to inflammation; however, certain SAAs (e.g., human SAA4) appear to be constitutively expressed or minimally induced in the inflammatory response.
Serum amyloid A proteins (SAA) comprise a superfamily of apolipoproteins produced in all vertebrates investigated to date (C. M. Uhlar, A. S. Whitehead, Serum amyloid A, the major vertebrate acute-phase reactant, Eur. J. Biochem. 265 (1999) 501-523). Depending on the species, three or four genetic loci that encode SAA have been identified and these genes are differentially expressed hepatically and/or extrahepatically (J. D. Sipe, Serum amyloid A: from fibril to function. Current status, Amyloid: Int. J. Exp. Clin. Invest. 7 (2000) 10-12). Acute phase serum amyloid A proteins (A-SAA) are predominately synthesized by the liver and are largely associated with the high-density lipoprotein 3 (HDL3) fraction of plasma (G. A. Coetzee, A. F. Strachan, D. R. Van Der Westhuyzen, H. C. Hoffe, M. S. Jeenah, F. C. De Beer, Serum amyloid A-containing human high density lipoprotein, J. Biol. Chem. 261 (1986) 9644-9651; N. Eriksen, E. P. Benditt, Isolation and characterization of the amyloid-related apoprotein (SAA) from human high density lipoprotein, Proc. Natl. Acad. Sci. USA 77 (1980) 6860-6864). Hepatically derived A-SAA levels can increase 1000-fold during the acute phase in response to the inflammatory cytokines IL-1, IL-6, and TNF-α (L. E. Jensen, A. S. Whitehead, Regulation of serum amyloid A protein expression during the acute-phase response, Biochem. J. 334 (1998) 489-503). The dramatic increase of A-SAA in circulation is achieved mainly by increased transcription (C. M. Uhlar, A. S. Whitehead, Serum amyloid A, the major vertebrate acute-phase reactant, Eur. J. Biochem. 265 (1999) 501-523).
The liver has been considered the primary site of SAA production. However, SAA production outside the liver has been found, on a limited basis. For instance, expression of SAA mRNA has been reported in human atherosclerotic lesions and in human cultured smooth muscle cells and monocyte/macrophage cell lines (Meek et al., 1994; Urieli-Shoval et al., 1994; Yamada et al., 1996), and a unique isoform of SAA (SAA3) is produced by rabbit synovial fibroblasts (Mitchell et al., J. Clin. Invest. 87: 1177-1185, 1991). More recently, it was discovered that SAA mRNA is widely expressed in many histologically normal epithelial tissues, including tissues of stomach, intestine, tonsil, breast, prostate, thyroid, lung, pancreas, kidney, skin and brain neurons (Urieli-Shoval et al., J. Histochem. Cytochem. 46: 1377-1384, 1998).
Experimental evidence from numerous investigators suggests a variety of functions for SAA proteins including suppression of immune responses (M. A. Aldo-Benson, M. D. Benson, SAA suppression of immune response in vitro: evidence for an effect on T cell-macrophage interaction, J. Immunol. 128 (1982) 2390-2392); inhibition of platelet aggregation (S. Zimlichman, A. Danaon, I. Nathan, G. Mozes, R. Shainkin-Kestenbaum, Serum amyloid A, an acute phase protein, inhibits platelet activation, J. Lab. Clin. Med. 116 (1990) 180-186); involvement in cholesterol/lipid metabolism (R. Kisilevsky, L. Subrahmanyan, Serum amyloid A changes high density lipoprotein's cellular affinity. A clue to serum amyloid A's principal function, Lab. Invest. 66 (1992) 778-785; R. L. Meek, N. Eriksen, E. P. Benditt, Murine serum amyloid A3 is a high density apolipoprotein and is secreted by macrophages, Proc. Natl. Acad. Sci. USA 89 (1992) 7949-7952); participation in detoxification of endotoxin (C. Baumberger, R. J. Ulevitch, J. M. Dayer, Modulation of endotoxic activity of lipopolysaccharide by high-density lipoprotein, Pathobiology 59 (1991) 378-383); induction of collagenase activity (C. E. Brinckerhoff, T. I. Mitchell, M. J. Karmilowicz, B. Kluve-Beckerman, M. D. Benson, Autocrine induction of collagenase by serum amyloid A-like and 2-microglobulin-like proteins, Science 243 (1989) 655-657); inhibition of neutrophil oxidative burst (R. P. Linke, V. Bock, G. Valet, G. To the, Inhibition of the oxidative burst response of N-formyl peptide-stimulated neutrophils by serum amyloid A protein, Biochem. Biophys. Res. Commun. 176 (1991) 1100-1105; M. E. Gatt, S. Urieli-Shoval, L. Preciado-Patt, M. Fridkin, S. Calco, Y. Azar, Y. Matzner, Effect of serum amyloid A on selected in vitro functions of isolated human neutrophils, J. Lab. Clin. Med. 132 (1998) 414-420); induction of migration of monocytes, polymorphonuclear leukocytes and T cells (R. Badaloto, J. M. Wang, W. J. Murphy, A. R. Lloyd, D. F. Michiel, L. L. Bausserman, D. J. Kelvin, J. J. Oppenheim, Serum amyloid A is a chemoattractant: induction of migration, adhesion and tissue infiltration of monocytes and polymorphonuclear leukocytes, J. Exp. Med. 180 (1994) 203-209; L. Xu, R. Badolato, W. J. Murphy, D. L. Longo, M. Anver, S. Hale, J. J. Oppenheim, J. M. Wang, A novel biologic function of serum amyloid A. Induction of T lymphocyte migration and adhesion, J. Immunol. 155 (1995) 1184-1190); and inhibition of cell adhesion to extracellular matrix components (L. Preciado-Patt, D. Levartowsky, M. Pras, R. Hershkoviz, O. Likder, M. Fridkin, Inhibition of cell adhesion to glycoproteins of the extracellular matrix by peptides corresponding to serum amyloid A. Toward understanding the physiological role of an enigmatic protein, Eur. J. Biochem. 223 (1994) 35-42). However, the primary physiological role of SAA in normal and disease states is not well understood.
Extrahepatic expression of human SAA mRNA and proteins has been demonstrated in macrophage, adipose, smooth muscle, and endothelial cells, suggesting a probable function at the site of production. Urieli-Shoval et al. determined that extrahepatic expression of human SAA was localized predominately to the epithelial components of a variety of tissues (S. Urieli-Shoval, P. Cohen, S. Eisenberg, Y. Matzner, Widespread expression of serum amyloid A in histologically normal human tissues: predominant localization to the epithelium, J. Histochem. Cytochem. 46 (1998) 1377-1384).
In humans there are four SAA genes clustered on chromosome 11p15.1 (G. C. Sellar, S. A. Jordan, W. A. Bickmore, J. A. Fantes, V. van Heyningen, A. S. Whitehead, The human serum amyloid A protein (SAA) superfamily gene cluster: mapping to chromosome 11p15.1 by physical and genetic linkage analysis, Genomics 19 (1994) 221-227). The hyperinducible SAA1 and SAA2 genes encode 104 residue A-SAA proteins that are 90% identical. SAA1 and SAA2 share approximately 95% overall nucleotide sequence identity in their promoter regions, exons, and introns (J. C. Betts, M. R. Edbrooke, R. V. Thakker, P. Woo, The human acute-phase serum amyloid A gene family: structure, evolution and expression in hepatoma cells, Scand. J. Immunol. 34 (1991) 471-482; P. Woo, J. Sipe, C. A. Dinarello, H. R. Colten, Structure of a human serum amyloid A gene and modulation of its expression in transfected L cells, J. Biol. Chem. 262 (1987) 15790-15795). Despite their sequence similarity, a recent study has demonstrated the differential glucocorticoid enhancement of SAA1 transcriptional expression compared to SAA2 in the context of cytokine-dependent induced expression (C. F. Thorn, A. S. Whitehead, Differential glucocorticoid enhancement of the cytokine-driven transcriptional activation of human acute phase serum amyloid A genes, SAA1 and SAA2, J. Immunol. 169 (2002) 399-406). Human SAA4, initially described by Betts et al. (J. C. Betts, M. R. Edbrooke, R. V. Thakker, P. Woo, The human acute-phase serum amyloid A gene family: structure, evolution and expression in hepatoma cells, Scand. J. Immunol. 34 (1991) 471-482), encodes constitutive SAA4 (C-SAA4) (D. M. Steel, G. C. Sellar, C. M. Uhlar, S. Simon, F. C. DeBeer, A. S. Whitehead, A constitutively expressed serum amyloid A protein gene (SAA4) is closely linked to, and share structural similarities with, an acute-phase serum amyloid A protein gene (SAA2), Genomics 16 (1993) 447-454; A. S. Whitehead, M. C. de Beer, D. M. Steel, M. Rits, J. M. Lelias, W. S. Lane, F. C. de Beer, Identification of novel members of the serum amyloid A protein superfamily as constitutive apolipoproteins of high density lipoproteins, J. Biol. Chem. 267 (1992) 3862-3867). In contrast to A-SAA1 and A-SAA2, C-SAA4 is not significantly induced during an acute phase response. C-SAA4 is 8 residues longer than A-SAA1 and A-SAA2 and shares only 55% identity with either A-SAA protein. C-SAA4 is present at low levels on both normal and acute phase HDL3, suggesting a probable housekeeping function for this protein.
The human SAA3 gene was initially identified by Sack and Talbot and was predicted to encode a 104 residue protein with 71% identity to A-SAA (G. H. Sack, C. C. Talbot, Jr., The human serum amyloid A (SAA)-encoding gene GSAA1: nucleotide sequence and possible autocrine-collagenase-inducer function, Gene 84 (1989) 509-515). However, a later genome-based study determined that a single nucleotide insertion within the predicted exon 3 would result in a truncated human SAA3 protein (B. Kluve-Beckerman, M. L. Drumm, M. D. Benson, Nonexpression of the human serum amyloid A three (SAA3) gene, DNA Cell Biol. 10 (1991) 651-661). To date, the SAA3 transcript or protein has not been detected in the human tissues or cell lines examined, nor has the region presumed to be the promoter of SAA3 been shown to be active (S. Urieli-Shoval, P. Cohen, S. Eisenberg, Y. Matzner, Widespread expression of serum amyloid A in histologically normal human tissues: predominant localization to the epithelium, J. Histochem. Cytochem. 46 (1998) 1377-1384); B. Kluve-Beckerman, M. L. Drumm, M. D. Benson, Nonexpression of the human serum amyloid A three (SAA3) gene, DNA Cell Biol. 10 (1991) 651-661; S. Urieli-Shoval, R. L. Meek, R. H. Hanson, N. Eriksen, E. P. Benditt, Human serum amyloid A genes are expressed in monocyte/macrophage cell lines, Am. J. Pathol. 145 (1994) 650-660; G. C. Sellar, A. S. Whitehead, Localization of four human serum amyloid A (SAA) protein superfamily genes to Chromosome 11p: Characterization of a fifth SAA-related gene sequence, Genomics 16 (1993) 774-776). These studies suggested that the SAA3 gene was either a pseudogene or the appropriate inducing conditions and/or cell type for SAA3 expression were not used.
Applicants have for the first time identified and demonstrated induced expression of the SAA3 gene and identified the transcript (cDNA) in human cells. It is an object of the present invention to provide the human SAA3 transcript and protein encoded thereby.
It is yet another object of the invention to provide nucleotide sequences which encode the human SAA3 protein.
It is yet another object of the invention to provide the amino acid sequence which comprises the human SAA3 protein.
It is yet another object of the invention to provide recombinant DNA protocols for using the sequences of the invention for production of recombinant SAA3, for use in assays to further delineate the role of SAA3 expression, for further understanding the acute phase immune response etc.
It is yet another object of the invention to provide assays for identifying inducers of SAA3 expression such as lipopolysaccharide (LPS) or prolactin (PRL) and to provide a promoter region capable of inducing expression in a human cell of operably linked sequences in the presence of these compounds.